Microoxic Niches within the Thylakoid Stroma of Air-Grown Chlamydomonas reinhardtii Protect [FeFe]-Hydrogenase and Support Hydrogen Production under Fully Aerobic Environment

Abstract

Photosynthetic hydrogen production in the microalga Chlamydomonas reinhardtii is catalyzed by two [FeFe]-hydrogenase isoforms, HydA1 and HydA2, both irreversibly inactivated upon a few seconds exposure to atmospheric oxygen. Until recently, it was thought that hydrogenase is not active in air-grown microalgal cells. In contrast, we show that the entire pool of cellular [FeFe]-hydrogenase remains active in air-grown cells due to efficient scavenging of oxygen. Using membrane inlet mass spectrometry, (18)O2 isotope, and various inhibitors, we were able to dissect the various oxygen uptake mechanisms. We found that both chlororespiration, catalyzed by plastid terminal oxidase, and Mehler reactions, catalyzed by photosystem I and Flavodiiron proteins, significantly contribute to oxygen uptake rate. This rate is considerably enhanced with increasing light, thus forming local anaerobic niches at the proximity of the stromal face of the thylakoid membrane. Furthermore, we found that in transition to high light, the hydrogen production rate is significantly enhanced for a short duration (100 s), thus indicating that [FeFe]-hydrogenase functions as an immediate sink for surplus electrons in aerobic as well as in anaerobic environments. In summary, we show that an anaerobic locality in the chloroplast preserves [FeFe]-hydrogenase activity and supports continuous hydrogen production in air-grown microalgal cells.

Figure 1.

Schematic illustration of the oxygen uptake mechanism in the chloroplast. Water splitting at PSII generates molecular oxygen, protons, and LEF. Electrons are transferred from PSII to the plastoquinone pool (Q_A; Q_B) from which the electron flow is transferred either to oxygen uptake in PTOX or to cytb₆f. From cytb₆f, electrons are transferred to plastocyanin (PC), which in turn reduces PSI that consumes oxygen directly via the Mehler reaction. The inhibitor DCMU specifically blocks electron output from PSII to the plastoquinone pool. Hence, upon addition of DCMU, the chloroplast oxygen uptake is omitted, without affecting mitochondrial respiration (not shown). The inhibitor DBMIB specifically blocks electron transfer output from cytb₆f, inhibiting oxygen consumption by the Mehler reaction, catalyzed by PSI, without affecting PTOX chlororespiration. n-PG is a specific inhibitor of PTOX.

Figure 2.

Photosynthetic activity and quantification of [FeFe]-hydrogenase in air-cultivated C. reinhardtii cells. A, Steady-state hydrogen production rates measured by MIMS in wild-type cultures cultivated under three light intensities: 77, 155, and 600 µE. Inset, Cells were grown in flasks stoppered with a sponge completely permeable to air, with constant light and stirring. B, LEF from PSII to hydrogen recorded upon transition from 77 µE to 1200 µE of either wild-type cells in the presence (broken green line) or absence (solid green line) of the PSII donor side inhibitor DCMU. The mutant hydEF-1 was used as negative control (black). C, Inhibition of hydrogenase by the inhibitor CO shuts down hydrogen production (green bar) in air-grown cells upon transition from 77 µE to 1200 µE at the onset of the recording. Photosynthetic oxygen production was not affected by the addition of CO (blue bars). D, Quantification of [FeFe]-hydrogenase in cells cultivated aerobically under 77, 155, and 600 µE. Top, An immunoblot performed using anti-HydA antibody. Purified [FeFe]-hydrogenase (5 ng of HydA) was used as marker and reference for band intensity quantification. Equal amounts of total protein were loaded in each lane. [FeFe]-hydrogenase quantities were normalized either to ng HydA per µg total protein (brown), shown in the left y axis, or to ng HydA per 1 million cells (orange), shown in the right y axis. The cultivation light intensities are shown at the bottom of each lane. All experiments were carried out in triplicates.

Figure 3.

[FeFe]-hydrogenase activity under fluctuating light in air-cultivated C. reinhardtii cells. A, The measured photosynthetic (green) versus chemical (orange) hydrogen production rates in cells cultivated aerobically (as shown in the inset of Fig. 2A) under varying irradiance. B, Recorded traces of net hydrogen (green) and oxygen (blue) kinetics as a function of irradiance. The top x axis shows the light intensity at each time point. Dissolved gases in the samples were measured simultaneously to illumination for 100 s under 77 µE (phase I), followed by a continuous illumination under 1200 µE. The onset of the transition to 1200 µE, lasting ∼100 S, where hydrogen production is maximal, is defined as phase II. The remaining period at 1200 µE is defined as phase III. C, Hydrogen production under fluctuating light. Photosynthetic hydrogen production rates during light irradiance of 77 µE punctuated each 2 min with 3 min of high irradiance (600, 1200, or 2400 µE). Phase II (yellow) and phase III (pink) are shown for each high light intensity. All experiments were carried out in triplicates. D, Pie graphs showing the divergence of oxygen consumption between the various mechanisms under the three phases shown in B. The percentages were calculated using the rates shown in Table I. Hence, while mitochondrial respiration (blue) was constant, 160 [µmol (O₂) mg⁻¹ h⁻¹], PTOX (green) and Mehler reactions (red) were significantly increased in a transition to high light.